During atherogenesis, alterations in eicosanoid biosynthesis occur by mechanisms not well understood. Prostaglandins (from arachidonic acid metabolism) and nitric oxide (7NO) produced in blood vessels are critical mediators in the regulation of vascular tone and inflammation. We, and others, have shown that 7NO modulates prostaglandin H2 synthase (PGHS, also known as cyclooxygenase) and alters eicosanoid production. Notably, the inducible forms of PGHS (PGHS-2) and nitric oxide synthase (iNOS) are increased and co-localize in atherosclerotic lesions and recent developments show that both enzymes bind. Our recent studies show that the lack of iNOS increases cellular PGHS-1 and PGHS-2 expression providing further evidence that link these pathways. Important milestones that we have reached include the finding that PGHS-1 is nitrated in human and murine atherosclerotic lesions and is dependent on iNOS. We believe that studies have neglected the role of iNOS in atherogenesis. Our central hypothesis is that nitrogen oxide species (NOx) regulate PGHS-1/-2 activity (by protein modification or through expression) and alters eicosanoid synthesis, which impacts on both atherogenesis and thrombosis. In Specific Aim 1, we will determine the role of iNOS in PGHS-dependent eicosanoid production during atherosclerosis and thrombosis. We propose that in the absence of iNOS, alterations in PGHS products occur shift the balance of prostacyclin (PGI2;anti-atherosclerotic/anti-thrombotic) and thromboxanes (TxA2;pro-atherosclerotic/pro-thrombotic) to favor inhibition of atherosclerosis. Using COX inhibitors, we will identify the specific roles of PGHS-1 and PGHS-2-derived eicosanoids in atherogenesis in ApoE-/- and ApoE-/-iNOS-/- mice. In mice lacking iNOS, we will define the role of PGHS and PGI2S-derived eicosanoids in the development of arterial thrombosis. In Specific Aim 2, studies will provide mechanisms that explain how eicosanoid synthesis is linked to iNOS and thus address in vivo and ex vivo observations from Aim 1. Thus, we propose to characterize NOx actions on arachidonic acid cascade enzymes and associated posttranslational modifications. Given that we identified significantly elevated levels of PGHS nitration in human and murine atherosclerotic lesions, we will identify sites of PGHS nitration and quantify extents of this modification in these tissues. Since we have revealed a heme-driven mechanism that leads to targeted Tyr385 nitration and PGHS inactivation, we will determine the role of this unique functionality in an atherosclerotic lesion. We will also investigate S-nitrosylation reactions of PGHS enzymes, which can provide a mechanism for enhanced eicosanoid production. Results from these studies will define the modulatory effects of NOx on PGHS function and eicosanoid production including the impact of these mediators on atherogenic and thrombotic processes and may highlight iNOS as a new target for therapeutic inhibition of inflammation.